Difference between revisions of "Part:BBa K5492203"
 
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<partinfo>BBa_K5492203 short</partinfo>
 
<partinfo>BBa_K5492203 short</partinfo>
  
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Optimised coding sequence of rat Diamine Oxidase appropiated for insertion into [https://parts.igem.org/Part:BBa_K5492101 pETDUET-1].
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Optimised coding sequence of Diamine Oxidase appropiated for insertion into [https://parts.igem.org/Part:BBa_K5492101 pETDUET-1]. The part was assembled from notable features of pETDUET-1, and [https://parts.igem.org/Part:BBa_K5492200 the optimised DAO gene].
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This part uses [https://parts.igem.org/Part:BBa_K5492100 BBa_K5492100] designed for the conservation of sequences during synthesis and PCR reactions.
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Adapter sequences from Twist Bioscience are added to the 5' and 3' end of the part.
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The primers for this sequence can be found here: [https://parts.igem.org/Part:BBa_K5492010 BBa_K5492010] [https://parts.igem.org/Part:BBa_K5492011 BBa_K5492011]
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===Usage and Biology===
 
===Usage and Biology===
 
Diamine oxidase (DAO) is an enzyme present in mammals, which plays an important role in digestion, as it is essential for the extracellular metabolism of histamine. DAO oxidizes histamine, and the resulting Imidazole-4-acetoldehyde isn’t able to bind to histamine receptors, therefore limiting the biological activity of histamine. Limiting the level of histamine effect can be an appropriate approach in cases of inflammation, where you can anticipate the histamine level increase. On our skin this includes some chronic diseases such as psoriasis, but more often would be useful in handling the acute increase of histamine level, such as a wasp bite. The decrease in oral histamine administration may also help to prevent some acute GI inflammation and smoothing the symptoms of some chronic bowel diseases.
 
Diamine oxidase (DAO) is an enzyme present in mammals, which plays an important role in digestion, as it is essential for the extracellular metabolism of histamine. DAO oxidizes histamine, and the resulting Imidazole-4-acetoldehyde isn’t able to bind to histamine receptors, therefore limiting the biological activity of histamine. Limiting the level of histamine effect can be an appropriate approach in cases of inflammation, where you can anticipate the histamine level increase. On our skin this includes some chronic diseases such as psoriasis, but more often would be useful in handling the acute increase of histamine level, such as a wasp bite. The decrease in oral histamine administration may also help to prevent some acute GI inflammation and smoothing the symptoms of some chronic bowel diseases.
  
 
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 +
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===Experiments===
 +
==PCR – optimized DAO==
 +
We have found the following PCR protocol the most optimal for the part:
 +
https://static.igem.wiki/teams/5492/registry/dao-pcr-plan.png
 +
 +
We added the following substrates in order: (each optimized and wild type)
 +
https://static.igem.wiki/teams/5492/registry/dao-pcr-substr.png
 +
 +
We added the 6X DNA Loading dye to each PCR tubes.
 +
We loaded the wells of the gels with 12μL of DAO solution in the following order: 
 +
 +
https://static.igem.wiki/teams/5492/registry/opt-dao-pcr.png
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1. D31O
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2. D32O
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3. D33O
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4. D34O
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5. Ladder 
 +
 +
==Restriction – optimized DAO==
 +
We performed digestion of our sequences using EcoRI and BglII.
 +
Our protocol:
 +
We added the following substrates in order:
 +
The used green buffer contained the loading dye too.
 +
 +
https://static.igem.wiki/teams/5492/registry/enzyme-restriction-substr.png
 +
 +
==Ligation==
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We added the following substrates in order:
 +
https://static.igem.wiki/teams/5492/registry/ligation-substr1.png
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==Transformation==
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Protocol for the transformation of DH10B E. coli strain
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1. For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
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2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA.
 +
Do not vortex.
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3. Place the mixture on ice for 30 minutes. Do not mix.
 +
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
 +
5. Place on ice for 5 minutes. Do not mix.
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6. Pipette 950 µl of room temperature SOC into the mixture.
 +
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
 +
8. Warm selection plates to 37°C.
 +
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
 +
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
 +
Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
 +
 +
After transforming the DH10B E. coli strain using the heat shock method, bacterial
 +
colonies successfully grew on the antibiotic-containing culture medium.
 +
https://static.igem.wiki/teams/5492/registry/transformation1.png
 +
 +
 +
==ELISA==
 +
 +
ELISA assay performed on HNMT/DAO proteins.
 +
 +
Table of measured absorbance at 450nm
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A1-A8: Standard stock, in 2X serial dilution.
 +
B1-B8 ; C1-C8; D1-D8: Capsuled enzyme with liposome, samples taken at different times.
 +
E1-E8; F1-F8; G1-G8: Referential enzyme without liposome, samples taken at different times.
 +
H1-H3: DAO in 1X; 5X; 10X dilution.
 +
 +
https://static.igem.wiki/teams/5492/registry/elisa/daoelisablue.png
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 +
 +
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https://static.igem.wiki/teams/5492/registry/elisa/daoelisa-table1.png
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 +
 
<partinfo>BBa_K5492203 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5492203 SequenceAndFeatures</partinfo>
  

Latest revision as of 12:54, 2 October 2024


Optimised DAO - pETDUET-1

Optimised coding sequence of Diamine Oxidase appropiated for insertion into pETDUET-1. The part was assembled from notable features of pETDUET-1, and the optimised DAO gene.

Usage and Biology

Diamine oxidase (DAO) is an enzyme present in mammals, which plays an important role in digestion, as it is essential for the extracellular metabolism of histamine. DAO oxidizes histamine, and the resulting Imidazole-4-acetoldehyde isn’t able to bind to histamine receptors, therefore limiting the biological activity of histamine. Limiting the level of histamine effect can be an appropriate approach in cases of inflammation, where you can anticipate the histamine level increase. On our skin this includes some chronic diseases such as psoriasis, but more often would be useful in handling the acute increase of histamine level, such as a wasp bite. The decrease in oral histamine administration may also help to prevent some acute GI inflammation and smoothing the symptoms of some chronic bowel diseases.


Experiments

PCR – optimized DAO

We have found the following PCR protocol the most optimal for the part: dao-pcr-plan.png

We added the following substrates in order: (each optimized and wild type) dao-pcr-substr.png

We added the 6X DNA Loading dye to each PCR tubes. We loaded the wells of the gels with 12μL of DAO solution in the following order: 

opt-dao-pcr.png 1. D31O 2. D32O 3. D33O 4. D34O 5. Ladder 

Restriction – optimized DAO

We performed digestion of our sequences using EcoRI and BglII. Our protocol: We added the following substrates in order: The used green buffer contained the loading dye too.

enzyme-restriction-substr.png

Ligation

We added the following substrates in order: ligation-substr1.png

Transformation

Protocol for the transformation of DH10B E. coli strain 

1. For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA.
Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

After transforming the DH10B E. coli strain using the heat shock method, bacterial colonies successfully grew on the antibiotic-containing culture medium. transformation1.png


ELISA

ELISA assay performed on HNMT/DAO proteins.

Table of measured absorbance at 450nm 

A1-A8: Standard stock, in 2X serial dilution.
B1-B8 ; C1-C8; D1-D8: Capsuled enzyme with liposome, samples taken at different times.
E1-E8; F1-F8; G1-G8: Referential enzyme without liposome, samples taken at different times.
H1-H3: DAO in 1X; 5X; 10X dilution.

daoelisablue.png


daoelisa-table1.png



Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 129
    Illegal XbaI site found at 47
    Illegal PstI site found at 190
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 129
    Illegal PstI site found at 190
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 129
    Illegal BglII site found at 2392
    Illegal BamHI site found at 123
    Illegal BamHI site found at 2067
    Illegal BamHI site found at 2321
    Illegal XhoI site found at 937
    Illegal XhoI site found at 2441
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 129
    Illegal XbaI site found at 47
    Illegal PstI site found at 190
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 129
    Illegal XbaI site found at 47
    Illegal PstI site found at 190
    Illegal NgoMIV site found at 2411
  • 1000
    COMPATIBLE WITH RFC[1000]